CN114438245B - SNP Molecular Markers Linked to the Major QTL Sites for Bacterial Wilt Resistance in Peanut and Its Application - Google Patents

Abstract

The invention relates to the technical field of molecular markers, in particular to a SNP molecular marker linked with the main QTL site of peanut resistance to bacterial wilt disease and its application. In the SNP molecular marker BWRB03-R provided by the present invention, the SNP polymorphism site is located at the 201st position of the sequence shown in SEQ ID NO.1, and the polymorphism is G/A. Specifically, in the SNP molecular marker BWRB03-R, the genotype of the polymorphic site is A, the corresponding peanut phenotype has strong bacterial wilt resistance, and the genotype of the polymorphic site is G, corresponding to The peanut phenotype is weaker bacterial wilt resistance. Using the SNP molecular marker provided by the invention can assist in the selection of bacterial wilt-resistant peanut materials, which is beneficial to promote the selection and breeding of bacterial wilt-resistant peanut varieties and improve the breeding efficiency.

Description

SNP Molecular Markers Linked to the Major QTL Sites for Bacterial Wilt Resistance in Peanut and Its Application

technical field

The invention relates to the technical field of molecular markers, in particular to a SNP molecular marker linked with the main QTL site qBWRB03 of peanut resistance to bacterial wilt disease and its application.

Background technique

Peanut (Arachis hypogea L.) is one of the important oil crops. The bacterial wilt disease caused by Rastonia solanacearum is the most important bacterial disease affecting peanut production. R. solanacearum invades the roots of plants from the soil, expands upward along the vascular bundles and proliferates, and eventually leads to the loss of water-carrying capacity of plant vessels and rapid withering and death. In peanut production, the mortality rate of plants in general diseased areas is 10-30%, and the mortality rate of plants in severely diseased areas can reach more than 80%. Therefore, effective prevention and control of bacterial wilt disease is one of the major demands for the development of the peanut industry.

Breeding and utilizing disease-resistant varieties is one of the most economical and effective measures to control peanut bacterial wilt. The resistance phenotype of peanut to bacterial wilt disease is quantitative resistance, and the discovery and utilization of stable major QTL is the key basis for the selection and breeding of bacterial wilt resistant varieties. By constructing a population of recombinant inbred lines, identifying bacterial wilt resistance in multiple environments, and performing QTL analysis combined with molecular marker genotypes, the major QTLs that are stably expressed can be identified; using molecular markers linked to QTLs can be used to identify major QTLs. Molecular marker-assisted selection to improve breeding efficiency.

At present, there are few studies on the QTL mapping of peanut resistance to bacterial wilt, and only the stable major QTL and its molecular markers have been discovered on the B02 chromosome. In order to further improve the resistance level of bacterial wilt, it is very important to discover and utilize new QTL major loci for disease resistance.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method for detecting the main QTL locus qBWRB03 of peanut resistance to bacterial wilt disease, and to quickly judge the bacterial wilt disease resistance of peanut.

Specifically, the first aspect of the present invention provides a peanut bacterial wilt resistance SNP molecular marker BWRB03-R. More specifically, the SNP molecular marker BWRB03-R provided by the present invention is linked to the main QTL site qBWRB03 for peanut resistance to bacterial wilt disease.

The SNP molecular marker BWRB03-R provided by the present invention is closely linked to the bacterial wilt resistance major QTL site qBWRB03 located on the B03 chromosome.

The SNP molecular marker provided by the present invention is BWRB03-R, and the 201st position of the sequence shown in SEQ ID NO.1 has a polymorphic site G/A.

In the SNP molecular marker BWRB03-R, the genotype with the 201st polymorphism site is A, which corresponds to the qBWRB03 containing the main QTL for bacterial wilt resistance, and the genotype is G, which corresponds to the absence of qBWRB03 .

The SNP molecular marker BWRB03-R provided by the present invention is amplified by using the primer pair shown in SEQ ID NO. 2-3.

In a second aspect, the present invention provides a primer pair, and the nucleotide sequence of the primer pair is shown in SEQ ID NO. 2-3. The primer pair provided by the present invention is used to amplify the above-mentioned SNP molecular marker BWRB03-R.

Primer pair for BWRB03-R:

Forward primer sequence 5'-ATGAAAAGTGAAAAATGAAAAGTGAAAAG GGA-3' (shown as SEQ ID NO.2),

Reverse primer sequence 5'-CAGCGGGGGCAAGTTCAC-3' (shown in SEQ ID NO. 3).

In a third aspect, the present invention claims a reagent or kit containing the above-mentioned primer pair.

According to the understanding of those skilled in the art, the present invention also claims to protect the above-mentioned SNP molecular marker BWRB03-R, or the primer pair shown in SEQ ID NO.2-3, or the primers containing SEQ ID NO.2-3 The application of the right reagent or kit in the detection of the main QTL locus qBWRB03 in peanut resistance to bacterial wilt.

Specifically, in the fourth aspect, the present invention provides a method for detecting the main QTL locus qBWRB03 of peanut resistance to bacterial wilt, comprising:

(1) using the primer pairs shown in SEQ ID NO.2-3 to carry out PCR amplification of the DNA of the peanut to be tested;

(2) Analyze the genotype of the SNP molecular marker BWRB03-R in the PCR amplification product, and judge whether the peanut to be tested contains the main QTL site qBWRB03 for resistance to bacterial wilt according to the genotype.

In the method for detecting the main QTL locus qBWRB03 for resistance to bacterial wilt in peanut provided by the present invention, if the genotype of the SNP molecular marker BWRB03-R is A, the peanut to be tested contains the main QTL locus qBWRB03 for resistance to bacterial wilt; The genotype of the SNP molecular marker BWRB03-R is G, and the peanuts to be tested do not contain qBWRB03.

The present invention also claims to protect the above-mentioned SNP molecular marker BWRB03-R, or the primer pair shown in SEQ ID NO.2-3, or a reagent or kit containing the primer pair described in SEQ ID NO.2-3, in Application of any of the following:

(1) Application in identification, improvement or molecular marker-assisted breeding of peanut germplasm resources;

(2) Application in early prediction of peanut bacterial wilt resistance;

(3) Application in screening peanuts with resistance to bacterial wilt.

A fifth aspect, the present invention provides a method for identifying bacterial wilt resistance in peanut, comprising:

(1) using the primers shown in SEQ ID NO.2-3 to carry out PCR amplification of the DNA of the peanut to be identified;

(2) Analyze the genotype of the SNP molecular marker BWRB03-R in the PCR amplification product, and determine the bacterial wilt resistance of the peanut to be identified according to the genotype.

In the method for identifying bacterial wilt resistance of peanuts provided by the present invention, if the genotype of the SNP molecular marker BWRB03-R is A, the peanut to be identified has strong bacterial wilt resistance; the genotype of the SNP molecular marker BWRB03-R is strong; For G, the resistance to bacterial wilt of the peanut to be identified is weak.

The beneficial effects of the present invention are:

The present invention obtains the SNP molecular marker BWRB03-R linked with the main locus qBWRB03 of resistance to bacterial wilt disease in peanut, and designs a primer pair for detecting the molecular marker of SNP, using the SNP molecular marker of the main locus qBWRB03 of resistance to bacterial wilt disease in peanut The detection method can assist in the selection of bacterial wilt-resistant peanut materials, which is beneficial to promote the selection and breeding of bacterial wilt-resistant peanut varieties and improve the breeding efficiency.

The SNP molecular marker of the present invention mainly detects whether it contains the bacterial wilt disease resistance site qBWRB03, and the peanut material containing the qBWRB03 site has stronger resistance to bacterial wilt disease. In breeding applications, ICG12625 or other materials with qBWRB03 can be used as parents for crosses, and many individuals with or without qBWRB03 will be produced in their progeny, and individual materials containing the qBWRB03 locus are retained through marker-assisted selection, and then from these If the individual materials are selected with high yield and good quality, new peanut varieties with resistance to bacterial wilt can finally be formed.

Description of drawings

Fig. 1 is the localization map of peanut bacterial wilt resistance major locus qBWRB03 on chromosome B03 in the present invention, wherein the vertical vertical line represents peanut chromosome B03, and the horizontal short line segment on the right side of B03 represents the genetic recombination bin on the chromosome; The short line segment represents the genetic distance (cM) between bins; the main locus of bacterial wilt resistance qBWRB03 is located in the 1cM region between the bin loci c13b013 and c13b014, and the right side of B03 lists the QTL detected in 2019-2020 to the confidence interval.

Detailed ways

The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are given for illustrative purposes only, and are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the spirit and spirit of the present invention.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1

This example provides the development process of the SNP molecular marker BWRB03-R linked to the major QTL site qBWRB03 for resistance to bacterial wilt in peanut, as follows.

Materials to be tested: crossed with the peanut germplasm susceptible to bacterial wilt disease Zhonghua 10 as the female parent and the peanut germplasm resistant to bacterial wilt disease ICG12625 as the male parent, and obtained a recombinant inbred line (RIL) containing 140 lines by single seed propagation. )group.

Phenotypic identification: In 2019 and 2020, the bacterial wilt resistance of 140 lines of Zhonghua 10, ICG12625 and RIL populations were identified in the Hong'an bacterial wilt disease nursery of the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences. A completely randomized block experimental design was used with 3 replicates. Each repetition of each material was planted with 20 plants in a row, with a row spacing of 30 cm and a plant spacing of 10 cm. Standard field management methods were adopted. After all seedlings emerged, the total number of plants in each material was counted. After the onset of bacterial wilt disease, the number of dead plants was counted until harvest. Then calculate the survival rate: (total number of plants - number of dead plants)/total number of plants × 100%, the higher the survival rate of materials, the better the resistance to bacterial wilt.

Construction of genetic linkage map: DNA from 140 individual leaves of parental and RIL populations was extracted by CTAB method; a 300-500bp Illumina Paired-end (PE) library was constructed for each sample DNA, and PE150 sequencing was performed. The raw data from the machine is quality controlled to obtain high-quality cleandata, and then BWA software is used to compare the cleandata to the reference genome of tetraploid cultivated peanut (https://www.peanutbase.org/data/public/Arachis_hypogaea/ Tifrunner.gnm1.KYV3/), and finally use the HaplotypeCaller module of GATK for SNP detection. The SNP sites with the same genotype were merged into bins, and finally the genetic map distance of bins was calculated to obtain the final linkage map. The linkage map contains 2701 bin sites, the total length is 1469.56cM, and the average distance between markers is 0.54cM.

Mapping of QTL: The QTL analysis of the survival rate was carried out using the interval mapping method of WinQTLCart software. The survival rate phenotype data of the two environments were all detected in the c13b013～c13b014 interval on chromosome B03. The main QTL locus qBWRB03, the main effect The LOD values of the QTL locus qBWRB03 in the two environments were 4.82 and 5.11, respectively, and the phenotypic interpretation rates were 12.01% and 10.58%, respectively.

The confidence intervals for the two environments overlapped each other and were located in a 1 cM interval (6.7–7.7 cM), a schematic diagram of which is shown in Figure 1. Therefore, the major locus of bacterial wilt resistance in peanut, qBWRB 03, can be stably expressed in the two-year experiment, and its resistance allele is derived from ICG12625.

A SNP molecular marker BWR B03-R (SEQ ID NO. 1) was developed by using a SNP in the interval c13b013～c13b014 (located on chromosome 125, 198, 196 bp (G/A variation) of the above reference genome B 03), and the amplified product was second-generation high The base composition of SNP sites was detected by flux sequencing: the A base was detected in ICG12625, which belonged to the allele containing qBWRB03 resistance to bacterial wilt; while the G base was detected in Zhonghua No. 10, it did not contain qBWRB03 anti-green wilt allele Blight allele.

For the molecular marker BWRB03-R provided in Example 1, the designed primers are shown in SEQ ID NO. 2-3.

Example 2

This example provides, using the SNP molecular marker BWRB03-R linked to the main locus of bacterial wilt resistance qBWRB03, for ICG12625 and 131 RILs of Zhonghua No. 10 for which survival rate data were obtained in both environments in Example 1 test. Specific steps are as follows.

Using genomic DNA as a template and the sequences shown in SEQ ID NO. 2-3 as primers, the SNP molecular marker BWRB03-R was amplified with the KAPA2G FastMultiplex Mix kit. PCR conditions were: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 15 s, renaturation at 55 °C for 30 s, extension at 72 °C for 30 s, a total of 30 cycles; final extension at 72 °C for 5 min, and incubation at 4 °C. After adding sequencing adapters to the amplified products, Paired-end 150bp (PE150) sequencing was performed on the Illumina HiSeq platform, and the sequenced sequences were compared to the reference sequences, and the bases of the SNP sites were detected. If the bases of RIL and the SNP site detected by the amplified fragment of ICG12625 are consistent, it indicates that the strain contains the bacterial wilt resistance allele of qBWRB03. At the same time, the results of the actually measured survival rate (see Example 1 for the test method) and the detection results of molecular markers were used for verification.

The results showed that among the 131 RILs, the SNP molecular marker genotype of 62 RILs was the same as that of ICG12625 (genotype classification 1, A), indicating that they contained a homozygous major anti-bacterial wilt QTL locus qBWRB03; The genotype of the SNP molecular marker is the same as that of Zhonghua No. 10 (genotype classification 2, G), indicating that it does not contain qBWRB03.

The T-test found that the 62 RILs containing qBWRB03 had significantly higher survival rates identified in both 2019 and 2020 than the 69 RILs that did not contain qBWRB03 (Table 1). Therefore, using the SNP molecular marker BWRB03-R linked with the main locus of bacterial wilt resistance qBWRB03 of the present invention to predict the resistance of peanut to bacterial wilt disease has a good prediction effect.

Table 1 Use the SNP molecular marker of the present invention to select the difference in survival rate of RIL strains

** Indicates a significant difference between genotype class 1 and genotype class 2 at the 0.01 level. The genotype of genotype classification 1 is the same as that of ICG12625, and the genotype of genotype classification 2 is the same as that of Zhonghua 10.

Although the present invention has been described in detail above with general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

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Claims (4)

1. The primer pair for amplifying the peanut bacterial wilt-resistant SNP molecular marker BWRB03-R is characterized in that the nucleotide sequence of the primer pair is shown as SEQ ID NO. 2-3; the SNP molecular marker BWRB03-R is a polymorphic site G/A at the 201 st site of a sequence shown in SEQ ID NO. 1.

2. A reagent or a kit comprising the primer set according to claim 1.

3. The primer pair of claim 1, or the reagent or kit of claim 2, for use in any one of:

(1) The application in early prediction of peanut bacterial wilt resistance;

(2) Application in screening of peanut with bacterial wilt resistance.

4. The method for identifying the resistance of peanut bacterial wilt is characterized by comprising the following steps:

(1) Carrying out PCR amplification on the DNA of the peanut to be identified by using a primer pair shown in SEQ ID NO. 2-3;

(2) Analyzing the genotype of the SNP molecular marker BWRB03-R in the PCR amplification product, and judging the bacterial wilt resistance of the peanut to be identified according to the genotype;

(3) If the genotype of the SNP molecular marker BWRB03-R is A, the bacterial wilt resistance of the peanut to be identified is strong; the genotype of the SNP molecular marker BWRB03-R is G, and the bacterial wilt resistance of the peanut to be identified is weak.

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